Pal et al., Oceanography 2013, 1:3
http://dx.doi.org/10.4172/2332-2632.1000111
Oceanography
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Evaluation of Relationship between Light Intensity (Lux) and Growth of
Chaetoceros muelleri
Sumilesh Wati Pal, Navneel Kunal Singh and Khairul Azam*
School of Marine Studies, Faculty of Science, Technology and Environment, University of the South Pacific, Fiji
Abstract
Microalgae have the most significant contribution towards the global environment, oxygen production and carbon
cycle consequently understanding marine microalgae is vital. Hence the factors that affect the growth should be
considered and studied in advance before the culturing of these algae. Photosynthesis is most important in plant
growth. Thus this study tries to find out the optimum light intensity (Lux) required for achieving maximum yield of
Chaetoceros muelleri in a laboratory culture.
The inoculation of the algae was done under controlled environment where the cells were counted and assumed
to be the same for all. The pure strand of C. muelleri with f2 media (8 sets of 3 flasks) were placed in front of the light
source at lux values of 0 lux, 500 lux, 1000 lux, 1500 lux, 2000 lux and 2500 lux. The cell count was the basic result that
was collected. At every 24 hours cells were counted and readings were recorded.
The experiment lasted 7 days. The result showed that there was significant growth that is the number of cells
increased exponentially as the Light intensity (lux) increased till the culture was exposed to a 1000 lux where maximum
cells were counted during the culture period (168 hours). From previous studies it was evident that the optimum light
intensity for flask culture in laboratory was 1000 lux. However, results from the experiment show that the cell growth with
1500 lux is very close and similar to 1000 lux. Some recommendations after the experiment are; that there is very little
literature on the above topic and that this area requires a lot more attention as Fiji’s Aquaculture Industries are blooming
thus there is a need for further research and study around the same area.
Keywords: Microalgae; Lux; Chaetoceros muelleri
Introduction
Microalgae are defined as minute photosynthetic plants and can
be found in both seawater and freshwater [1]. These have similar
properties as land based plants but they have to be submerged in an
aqueous environment where they have efficient access to water, carbon
dioxide and other nutrients.
Nowadays, there are several marketable applications of microalgae;
some of which include (i) they are used to enhance the nourishing
value of food and animal feed owing to their chemical composition, (ii)
they play a crucial role in aquaculture and (iii) they can be assimilated
into cosmetics. Moreover, they are cultivated as a source of highly
valuable molecules [2]. Microalgae are also used in the production
of pharmaceuticals, diet supplements, pigments and biofuel [3] and
represent the largest, yet one of the most poorly understood groups of
microorganisms on earth [4]. A handful of microalgae species out of
the many that have been found are mainly cultured for live aquaculture
feed and Chaetoceros muelleri [5] is one of them.
The hypothesis for this experiment is to find whether light is
required to culture Chaetoceros muelleri. Secondly to examine the
amount of light required to give the maximum yield of the product.
Also show the relationship between the amount of light required and
the growth rate of Chaetoceros muelleri.
Microalgal culture has to be conducted under specialised
conditions as explained by Coutteau [6]; the most important
parameters regulating algal growth are nutrient quantity and quality,
light, pH, turbulence, salinity and temperature. Concentrations of cells
in phytoplankton cultures are generally higher than those found in
nature. Algal cultures must therefore be enriched with nutrients to
make up for the deficiencies in the seawater. Macronutrients include
nitrate, phosphate and silicate. Here the paper describes the general
Oceanography
ISSN: 2332-2632 Oceanography, an open access journal
set for culturing Microalgae: Temperature 16-27°C, Salinity 12-40g/l
and Light Intensity (lux) 1,000-10,000 lux depending on the volume
of culture.
As with all plants, microalgae photosynthesize, i.e., they assimilate
inorganic carbon for transformation into carbon-based matter. Light
is the source of energy which drives this reaction and in this regard
intensity, spectral quality and photoperiod need to be considered. Light
intensity plays an vital role, but the necessities fluctuate critically with
the culture penetration and the density of the algal culture: at higher
depths and cell concentrations the light intensity must be increased
to penetrate through the culture 1,000 lux is suitable for Erlenmeyer
flasks, 5,000-10,000 lux is required for larger volumes [6]. Light may be
natural or supplied by fluorescent tubes. Too high light intensity (e.g.
direct sun light, small container close to artificial light) may result in
photo-inhibition. Also, overheating due to both natural and artificial
illumination should be avoided. The duration of artificial illumination
should be minimum 18h of light per day, although cultivated
phytoplankton develops normally under constant illumination [7].
Background
Chaetoceros is probably the largest genus of marine planktonic
*Corresponding author: Khairul Azam, School of Marine Studies, Faculty of
Science, Technology and Environment, University of the South Pacific, Fiji, Tel:
+679 8341915; E-mail: azam_k@usp.ac.fj
Received July 06, 2013; Accepted July 18, 2013; Published July 22, 2013
Citation: Pal SW, Singh NK, Azam K (2013) Evaluation of Relationship between
Light Intensity (Lux) and Growth of Chaetoceros muelleri. Oceanography 1: 111.
doi:10.4172/2332-2632.1000111
Copyright: © 2013 Pal SW, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Volume 1 • Issue 3 • 1000111
Citation: Pal SW, Singh NK, Azam K (2013) Evaluation of Relationship between Light Intensity (Lux) and Growth of Chaetoceros muelleri.
Oceanography 1: 111. doi:10.4172/2332-2632.1000111
Page 2 of 4
diatoms with approximately 400 species described. Although a large
number of these descriptions are no longer valid. It is often very difficult
to distinguish between different Chaetoceros species. Several attempts
have been made to restructure this large genus into subgenera and this
work is still in progress. However, most of the effort to describe species
have been focused in boreal areas, and the genus is cosmopolitan [7], so
there are probably a large number of tropical species still undescribed.
National Algae Culture Collection (ANACC), CSIRO in Tasmania,
Australia with the assistance of the USP Laboratory Technician.
Glassware preparation
The glassware used in this experiment was cleaned and washed
with detergent and autoclaved on programme A (121°C and 20 psi) as
provided on the autoclave for 15 minutes.
Scientific Classification of Chaetoceros muelleri:
Water treatment
Empire Eukaryota
Kingdom Chromista
Subkingdom Harosa
Infrakingdom Heterokonta
Phylum Ochrophyta
Subphylum Khakista
Class Bacillariophyceae
Subclass Coscinodiscophycidae
Superorder Chaetocerotanae
Order Chaetocerotales
Family Chaetocerotaceae
Genus Chaetoceros
Seawater was strained using 1 µm (micro meter) cartridge filter
and used for the preparation of the Media and Stock solution. After
filtration the water was passed a Life Guard brand Ultra Violet Steriliser
(Model no. - QL 40) that uses a 40 watt UV sterilizer bulb. All the above
was done in the Aquaculture Laboratory (Wet Lab).
In the past there have been very few studies done on the relationship
between the lux and the growth rate of Chaetoceros muelleri. As stated
by Blinn [8] respiration and photosynthesis are the two significant
processes in microalgal growth that occur simultaneously in the
light. To know the rates of both processes, at least one of them has
to be measured. To be able to measure the rate of light respiration of
Chlorella sorokiniana, the measurement of oxygen uptake must be fast,
preferably in the order of minutes. This experiment looks deep into
the process of Respiration where the amount of Oxygen uptake in situ.
The respiration rate is known to be related to the growth rate, and it is
suggested that faster algal growth leads to a higher energy requirement,
and as such, respiratory activity increases [8].
And as stated by Brown [9] equal inocula were added in the two
bioreactors (1 and 3 cm light path); by the end of the second day, cell
concentration was low in the 1-cm light path bioreactor, possibly
indicating photo inhibition, since growth of the culture increased in
the 3-cm light path bioreactor.. The optimal population density was
obtained by a daily harvest of 5% of culture volume to be 2.39 gm−2
day−1 in 1 cm light path and 3.27 gm−2 day−1 in 3 cm light path. Light
path length of 3 cm was found optimal in both with low and high initial
cell densities [10].
As noted by the previous studies there has not been much research
done on Chaetoceros muelleri in regards to the light and growth of
the microalgae. Plus this research proposals will be one of the pioneer
projects that will consider the relationship for the two variables in the
labs as small experiment, from where more research can be carried
out in order to use the investigate the intensity of light required for
the maximum growth of Chaetoceros muelleri. Hence there is more
attention required for the above pioneer project to be carried out
successfully.
Materials and Methods
The experiment was carried out in the laboratory of the School of
Marine Studies, University of the South Pacific (USP), Suva, Fiji.
Experimental organisms (C. muelleri)
A pure 20 ml sample of C. muelleri was purchased from Australian
Oceanography
ISSN: 2332-2632 Oceanography, an open access journal
F/2 media preparation and modification
The procedures used to prepare the F/2 Media and also the
Modification of F/2 media were followed from Guillard [11]. 75 g of
NaNO3 and 5g of NaH2PO4 H2O were dissolved in 1 litre of filtered sea
water and this was labelled as Solution A. Trace Metals was labelled
Solution B, Silicates was labelled solution C. 1 ml of solution B and C
was used for every litre of media. Vitamins were labelled Solution D
and 0.5 ml of this solution was used for every litre of media.
Inoculation
A sample of pure C. muelleri is placed in a controlled environment
for it to grow or reproduce. For this experiment the procedure used
was adapted from Probert and Klaas [12] was followed. 100 ml of
the f/2 media that was prepared earlier was filled into 250 ml Conical
culture flasks which were covered using corks and aluminium foil.
The prepared flasks were autoclaved under Programme A. After the
Autoclave the flasks were removed and left overnight to cool in the
algae room. All the flasks were inoculated with 10% of inoculum (C.
muelleri) in the Laminar Flow cupboard. The cabinet was cleaned with
70% ethanol. The pippttes and micro pipettes were also autoclaved
[13]. The Nitrile hand gloves used are to be sprayed with 70% ethanol
and will be worn while inoculating. The mouth of the culture flask was
sterilised by moving over a Bunsen burner flame.
Experimental setup
After the inoculation the rubber corks were bored to make holes
and the glass tubing were inserted for the inlet (3-4 mm) and exhaust
(2-3 mm) were placed to seal the mouth. The above setup was prepared
in the laminar flow cabinet to avoid contamination. Once the inlet and
exhaust lines are fixed the flasks were ready for setup in the algae room.
Some of the parameters that were kept constant were; temperature at
18°C, preferred pH of 7-9 with reference to FAO, Instrumental Paper,
1999, salinity was between 20-35 ppt, Aeration was controlled by
opening or closing the switch valves linked to the inlet line and the
cell starter culture the cells were calculated in terms of cells/ml. the
inoculation required 10% of the starter culture. During the preparation
of all the twelve flasks efforts were made to ensure that approximately
the same number of cells is transferred.
The variable
The variable in the experiment is the lux that is a standardized
unit of measurement of the light intensity (which can also be called
“illuminance” or “illumination”) - as an example for reference
purposes. One lux is equal to one lumen per square metre [14]:
Volume 1 • Issue 3 • 1000111
Citation: Pal SW, Singh NK, Azam K (2013) Evaluation of Relationship between Light Intensity (Lux) and Growth of Chaetoceros muelleri.
Oceanography 1: 111. doi:10.4172/2332-2632.1000111
Page 3 of 4
1 lx = 1 lm/m2 = 1 cd·sr·m–2
The flasks were placed at 500 lux, 1000 lux and at 1500 lux from
the light source. The growth performance was measured in terms of
cell density/ml.
Experimental design
Eighteen (18) trails were carried out that is six treatments with
three replicates of each. The six treatments were based on the different
lux; 0 lux, 500 lux, 1000 lux, 1500 lux, 2000 lux and 2500 lux. These
were represented as treatment A, B, C, D, E and F respectively.
Sampling
Hemocytometer was used to count the cells [2]. From the time the
inoculation was done the counting was done at every 24 hour time
interval until the culture reached a stationary stage. From every flask
a sample of 1 ml was taken and average of 3 counts were done for
better result analysis. The experiment was repeated two more times to
confirm and reconfirm results that were obtained in the first set.
Statistical analysis
The statistical analysis that was adapted by this experiment was
SPSS. This showed the relationship between the amount of flux and the
growth rate at different lux rates. The comparison of the differences of
the treatments with the control was done by the Duncan’s Test.
Results
Over the period of the experiment the results were collected and
tabulated as Replicate 1, Replicate 2 and Replicate 3 (Table 1) from
which an average data was calculated. Table 1 shows the average of all
the three Replicates that was done. The values at the different lux (0
lux, 500 lux, 1000 lux, 1500 lux, 2000 lux and 2500 lux) were added and
divided by 3.
Average Cell Count at Different Lux =
Cell count T1 + Cell Count T2 + Cell Count T3
3 Treatments
More over Figure 1 was constructed using Table 1, in order to see
the relationship between the Cell counts at different lux with the time
of each count.
According to the average growth rate of the C. muelleri, the figure
states that the highest cell count is at 1000 lux. From the 0 hour at
stated lux the cell count increased gradually. The maximum growth is
shown at 1000 lux around 96 hours. As indicated by the figure that
a maximum growth is reached than the slows and starts to decrease.
This figure indicates the phases exponential phase (growth phase),
stationary phase (Maximum point reached) than the death phase (no
Average Cell Count
Cell Count (x10 ^6)
Time
(Hrs)
0 Lux
500 Lux
1000 Lux 1500 Lux 2000 Lux
2500 Lux
0
0.2
0.296667
0.2
0.2
0.2
0.2
24
0.126667
0.723333
1.14
1.04
0.95
0.78
48
0.03
1.463333
2.28
1.6333
1.636667
1.42
72
0.005667
2.416667
3.41
2.8033
2.586667
1.973333
96
0
2.876667 3.776667
3.2433
2.873333
2.686667
120
0
3.196667 3.606667
3.0667
2.803333
2.973333
144
0
2.826667 3.596667
3.1133
2.823333
2.863333
168
0
2.483333 3.063333
2.9233
2.74
2.776667
Table 1: The average cell count for the three treatments.
Oceanography
ISSN: 2332-2632 Oceanography, an open access journal
more growth). At all the different lux (0 lux, 500 lux, 1000 lux, 1500 lux,
2000 lux and 2500 lux) there is a similar trend seen. The control (0 lux)
showded little growth initially, than the cell count decreased to zero.
Moreover from the SPSS Statistics that was carried out indicated
that there was a significant (df5, F298.54, p 0.00) difference between
6 treatments (0 lux, 500 lux, 1500 lux, 2000 lux and 2500 lux). 1000
lux was significantly higher than the control (p 0.00), 2000 lux (p 0.00)
AND 2500 lux (p 0.00), however it was not significantly different from
500 lux (p 0.899) and 1500 lux (p 0.221). Thus it can be noticed that
1000 lux is more similar to 1500 lux. Figure 1 shows that at 1000 lux
there is highest growth.
Discussion
The purpose if this experiment was to better understand the
importance of light intensity in the growth of the C. muelleri as the time
and the intensity was increased. The experiment was modelled in such
a way that at different lux from a minimum to a maximum lux just to
compare the cell count to observe the cell growth, the control was also
set up to witness if there will be any growth at zero light intensity. The
purpose of having the different lux setup for the experiment was to find
out the best light intensity required for the Flask culture of C. muelleri
in a controlled laboratory.
The main result of the experiment showed that growth rate varied
at different Light intensity (Lux). C. muelleri can be cultured in a
laboratory at different light intensity but according to the results the
best amount of light for the maximum yield is 1000 lux, 500 lux and
also 1500 lux are also similar but lower growth rate seen than 1000 lux.
The experiment results is supported by Coutteau [6] where
he stated that Light intensity played an important role, but the
requirements varied greatly with the culture depth and the density
of the algal culture: at higher depths and cell concentrations the light
intensity must be increased to penetrate through the culture (e.g. 1,000
lux is suitable for Erlenmeyer flasks, 5,000-10,000 is required for larger
volumes). Too high light intensity (e.g. direct sun light, small container
close to artificial light) may result in photo-inhibition. This may cause
overheating due to both natural and artificial illumination [15]. Thus in
this experiment states that the higher the lux the slower the growth rate
at around 2000 lux and 2500 lux the growth fast initially that it slows;
there might be few factors affecting this one could be the light intensity
too high for the cells to tolerate, or the high population increase might
decrease the nutrients thus slow growth rate. Hence this indicates that
too much light is not suitable for the growth of C. muelleri. Comparing
this to 500 lux; this also has slow growth rate; thus only conclusion that
can be drawn is less light intensity induces slow growth rate, as the flask
has enough nutrients, light was the only factor that was low.
Naturally not only Light is required for the microalgae growth, there
are other factors that affect the growth rate of C. muelleri which include
right salinity, right pH, right amount of nutrients and vitamins [16].
The term nutrient “limitation” has been widely used in the literature
in a variety of contexts [17], often interchangeably with the terms
“deficiency” and “starvation.” There are two distinctively different types
of limitation: one referring to limitation of the maximum population
density achieved (Liebig limitation) and one referring to limitation of
growth rates
From the earlier research’s carried out for the laboratory culture
of C. muelleri, the light intensity required should be 1000 lux and
the results for this experiment showed a similar outcome along with
Volume 1 • Issue 3 • 1000111
Citation: Pal SW, Singh NK, Azam K (2013) Evaluation of Relationship between Light Intensity (Lux) and Growth of Chaetoceros muelleri.
Oceanography 1: 111. doi:10.4172/2332-2632.1000111
Page 4 of 4
3. Creswell L (2010) Phytoplankton Culture for Aquaculture Feed. Southern
Regional Aquaculture Center.
The Graph of Cell Count vs Time at Standard Lux
4
4. Guedes AC, Malcata FX (2012) Nutritional Value and Uses of Microalgae in
Aquaculture. Agricultural and Biological Sciences.
3.5
Cell Count (*10^6)
3
0 LUX
2.5
500 LUX
1000 LUX
2
1500 LUX
1.5
2000 Lux
1
2500 Lux
0.5
0
-0.5
0
24
48
72
96
TIME (hours)
120
144
168
Figure 1: The Average cell growth for C. muelleri over a period of 168hrs and
at the stated Lux.
the other necessary factors that were kept constant. This experiment
showed the effect of light on the growth of C. muelleri which was only
one factor. There are further studies required in the similar areas. Once
there is a better understanding of the culture of microalgae at a small
scale, this could be used in the mass culture and used in the developing
aquaculture industry in Fiji and the Pacific Islands.
Conclusion
This study examined the amount of light intensity required to give
the best yield of C. muelleri in a laboratory culture. From the experiment
carried out and the data analysis it may be concluded that the 1000 Lux
is the optimum lux to produce the best yield. This is supported by the
literature. It was noticed that as light intensity increased the growth of
C. muelleri increased significantly to a limit at 500 lux, 2000 lux and
2500 lux where the growth rate slowed. This could be due to the either
the decrease in the nutrient level in the flask or due to too less or over
heat of the flask.
The relationship between light intensity required and the growth
rate of Chaetoceros muelleri was also studied (Figure 1). Light is
required for the growth of microalgae on the other hand too much
light and too little light can slow the growth rate [17,18]. Thus the
experiment indicated that 1000 lux to 1500 lux is the best light intensity
in order to produce maximum yield.
5. Raghvan G, Haridevi CK (2008) Growth and proximate composition of the
Chaetoceros calcitrans f. pumilus under different temperature, salinity and
carbon dioxide levels. Aquaculture Research 39: 1053-1058.
6. Coutteau P (1996) Manual on the production and use of live food for
aquaculture. FAO Fisheries Technical Paper 361.
7. Hasle GR, Syvertsen EE (1997) Marine Diatoms. Identifying Marine
Phytoplankton. Academic Press, San Diego, USA.
8. Blinn DW (1984) Growth responses to variations in temperature and specific
conductance by Chaetoceros muelleri (Bacillariophyceae). Brit Phycol J 19:
31-35.
9. Brown MR (2002) Nutritional Value and Use of Microalgae in Aquaculture.
Quintana Roo, Cancún México.
10.Alabi AO, Tampier M, Bibeau E (2009) Microalgae Technologies for and
processes for biofuel/ bioenergy in British Columbia: Current Technology,
Suitability & Barriers to Implementation. The British Columbia Innovation
Council.
11.Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates.
Culture of Marine Invertebrate Animals, Springer, USA.
12.Probert I, Klaas C (1999) Microalgal culturing. Practical notes from the culturing
short course.
13.Thornton DCO (2009) Effect of low ph on carbohydrate production by a marine
planktonic diatom (Chaetoceros muelleri). Research Letters in Ecology.
14.Hoff FH, Snell TW (2008) Plankton Culture Manual. Florida Aquafarms, Florida,
USA.
15.Whitton BA, John DM, Johnson LR, Boulton PNG, Kelly, MG, et al. (1998) A
coded list of freshwater algae of the British Isles.
16.Leonardos N, Geider RJ (2004) Responses of Elemental and Biochemical
Composition of Chaetoceros muelleri to Growth under Varying Light and
Nitrate: Phosphate Supply Ratios and Their Influence on Critical N:P.
17.Beardall J, Young E, Robert S (2001) Approaches for determining phytoplankton
nutrient limitation. Aquat Sci 63: 44-69.
18.Ihnken S, Roberts S, Beardall J (2011) Differential responses of growth and
photosynthesis in the marine diatom Chaetoceros muelleri to CO2 and light
availability. Phycologia 50: 182-193.
Acknowledgement
The authors wish to thank Mr. Jone Lima and Mr. Ravikash Prasad for their
assistance in getting the pure sample of C. mulleri from Australia and assisting in
Statistical Analysis using SPSS software respectively. The authors are grateful to
Pacific Islands Forum Secretariat for providing financial assistance in the project
and research.
References
1. Carlsson AS, Beilen J, Möller R, Clayton D (2007) Micro- and macro-algae:
utility for industrial applications. CPL Press.
2. Polkinghorne C (2010) Procedure for Assessing Chronic Residual Toxicity
of a Ballast Water Treatment System to the Green Alga (Selenastrum
capricornutum). Standard Operating Procedure.
Citation: Pal SW, Singh NK, Azam K (2013) Evaluation of Relationship between
Light Intensity (Lux) and Growth of Chaetoceros muelleri. Oceanography 1:
111. doi:10.4172/2332-2632.1000111
Oceanography
ISSN: 2332-2632 Oceanography, an open access journal
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